Empirical methods

Graded supplementation technique

The empirical method most commonly used to determine amino acid responses in growing poultry involves the addition of graded supplements of the amino acid under test to a basal diet deficient in that amino acid (D'Mello, 1982). These graded additions are accomplished with the crystalline form of the amino acid. A number of criteria must be fulfilled for satisfactory results. It is imperative that the basal diet is sufficiently deficient in the amino acid under investigation and that graded doses of the pure amino acid are employed to generate a smooth and full response curve encompassing both growth-limiting doses and those that elicit a maximum response. It follows that data derived from supplementation experiments involving just one or two additions or those from bioassays which lack maximum response values are unsuitable for assessing optimal economic doses. In certain instances it may be necessary to use different combinations of those amino acids involved in antagonistic relationships. The measurement of food intake is essential for complete interpretation of data. Providing these criteria are satisfied, the response curve may be used to estimate optimal doses for given rates of growth or food efficiency. In addition, the response curve may be used to determine estimates of slope and plateau values required as important components for the Reading model (Fisher, 1994).

Diet-dilution technique

Fisher and Morris (1970) proposed an alternative empirical method based on the sequential dilution of a high-protein 'summit' diet with an isoenergetic protein-free mixture. This method has been described at length in Chapter 7 since the technique relies on the deliberate creation of an imbalance in the summit diet. The imbalance is accomplished by maintaining large dietary excesses of all essential amino acids except the one under test, which is fixed at a markedly lower level.

Limitations

Neither method is without its limitations. In particular, the graded supplementation technique has been criticized on several counts (Fisher and Morris, 1970; Gous, 1980). Firstly, it is claimed that that dietary amino acid balance changes with each successive dose of the limiting amino acid and that the response may be influenced by this factor. The exact mechanism by which changing amino acid balance might affect the response has never been amplified by the critics. Sines any change in amino acid balance would affect food intake rather than the efficiency of utilization of the limiting amino acid (see Chapter 7), the first criticism may be discounted. It is also argued that at high levels of supplementation, the amino acid under test may no longer be first-limiting and that further responses to that amino acid might be prevented by the second-limiting amino acid(s). An additional criticism centres on the alleged difficulty in devising a suitable basal diet which is sufficiently deficient to allow the use of a wide range of input levels of the limiting amino acid. The final criticism relates to the cost of certain synthetic amino acids which, Gous (1980) maintains, might prevent supplementation studies with the more expensive amino acids.

It is argued that none of the disadvantages just described apply to the diet-dilution technique. Indeed, Fisher and Morris (1970) claimed that this technique satisfied 'all the requirements for a successful assay' and Gous (1980) described it as an 'improved method'. These assertions imply that the diet-dilution technique is capable of yielding more valid response data than the graded supplementation method but no evidence has yet emerged to substantiate this claim.

It is instructive to recall that although the initial description of the diet-dilution technique appeared over 30 years ago, the graded supplementation procedure still remains the method of choice in the vast majority of studies on the amino acid responses of growing poultry (Hewitt and Lewis, 1972; Boomgaardt and Baker, 1973; D'Mello and Emmans, 1975; Edwards and Baker, 1999; Tesseraud et al., 1999). It is clear that the cost of amino acids has not been a major deterrent to this research where the primary costs are likely to be capital expenditure and labour. The other criticisms levelled at the graded supplementation technique may also be rejected. In particular, it is possible to discount the assertion that other limiting amino acids may inhibit the maximum response to the first-limiting amino acid. It is now common practice to include generous levels of protein in the diet, and by judicious supplementation it should be possible to ensure adequacy of all other amino acids. In addition, the use of combined supplements of the first- and second-limiting amino acids may be employed (D'Mello and Lewis, 1970; D'Mello and Emmans, 1975). Such an approach has yielded satisfactory responses to arginine and lysine in turkey poults (D'Mello and Emmans, 1975), with maximum growth rates comparable to those observed in groups fed a standard diet. Furthermore, in many studies a wide range of input levels of the first-limiting amino acid have been ensured (Hewitt and Lewis, 1972; Boomgaardt and Baker, 1973; D'Mello and Emmans, 1975; D'Mello, 1990). Thus there appears to have been little difficulty in devising suitably deficient basal diets or in providing a satisfactory range of intakes of the limiting amino acid. The most serious criticism levelled at the graded supplementation technique relates to the systematic variation in amino acid balance of successive diets within a supplementation series. However, it will be apparent from the account in Chapter 7 that the diet-dilution technique relies on the deliberate imposition of an amino acid imbalance in the summit diet by the provision of all amino acids, except that under test, in substantial excess. This is the precise method for the precipitation of deleterious effects in amino acid imbalance (Harper, 1964; D'Mello and Lewis, 1971; D'Mello, 1990).

D'Mello (1982) stated that although it is relatively straightforward to generate response curves to a single amino acid by the diet-dilution technique, considerable difficulties may emerge when responses to interacting pairs such as lysine and arginine or leucine and valine are to be determined. It was suggested that the responses to arginine or valine would be unreliable owing to the constraint to design the summit diet with large excesses of other amino acids including lysine and leucine. The adverse effects of lysine and leucine are well documented and will be addressed later in this chapter. Gous (1980), however, argues that this issue is readily resolved by formulating a number of dilution series each containing different concentrations of the antagonizing amino acid. The degree of interaction would be indicated by the resultant difference in slope of individual response curves across the series.

Comparison of techniques

In a direct comparison of the two methods, D'Mello (1982) used data obtained by the diet-dilution procedure (Gous, 1980) and by the graded supplementation technique (Boomgaardt and Baker, 1973) with respect to growth responses of broiler chicks to varying lysine intakes (Fig. 14.1). The data of Stockland et al. (1970), obtained with rats fed graded supplements of lysine, were also

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0 100 200 300 400 500 600

Lysine intake (mg day-1)

Fig. 14.1. Daily weight gain of growing rats and chicks in relation to daily lysine intake. (From D'Mello, 1982; source of data: rats (•), Stockland et al., 1970; chicks (▲), Boomgaardt and Baker, 1973; chicks (A), Gous, 1980. Reproduced with permission of The World's Poultry Science Association.)

incorporated in this figure. It became clear that when growth increments were considered in relation to daily lysine intake both methods yielded similar results with all 'limiting points' contributing to a single response curve. In a subsequent examination of data obtained with turkey poults, D'Mello (1983) confirmed the high degree of compatibility between the two methods. If the diet-dilution technique is a superior method, then the responses generated by the supplementation procedure would be displaced positively with respect to the data of Gous (1980). The agreement between the rat and chick data, irrespective of the method used to elicit responses, is noteworthy. Such interspecies compatibility in amino acid response data is reviewed later in this chapter, but it should be recognized here that these similarities were obtained with the graded supplementation technique despite wide differences in dietary amino acid balance and in the nature of protein ingredients used. On the basis of the evidence presented in Fig. 14.1, D'Mello (1982) concluded that the lack of confidence in the graded supplementation technique and the projection of the diet-dilution procedure as an improved method could not be justified since both methods yielded concordant growth responses. Boorman and Burgess (1986) arrived at a similar conclusion after a detailed analysis of 15 data sets derived by the supplementation technique and 11 data sets obtained by the diet-dilution method in studies on lysine responses in broiler chicks.

Since the reviews of D'Mello (1982) and Boorman and Burgess (1986), a disquieting feature of the diet-dilution technique has emerged with far-reaching consequences which question the authenticity of earlier attempts to validate the technique (Gous, 1980; Gous and Morris, 1985). It is a condition of this procedure that growth responses are not confounded by the deliberate variation in dietary CP content which occurs on dilution of the summit diet. The method relies on the interpretation of responses to different rates of dilution as responses to the first-limiting amino acid and not to changing dietary CP contents. In this particular respect, Gous and Morris (1985) confirmed that the response in gain when lysine was added to diets in the dilution series 'corresponded almost exactly to the level of lysine in the diet, irrespective of protein content'. Indeed, multiple regression analysis of the data indicated that CP intake did not contribute significantly to the best-fit model. Gous and Morris (1985) proceeded to question the need to use isonitroge-nous diets when determining responses of growing poultry to individual amino acids. However, in subsequent and more detailed investigations with chicks, these author« (Morris et al., 1987; Abebe and Morris, 1990a,b) demonstrated that CP level unequivocally influenced the growth response to an amino acid. The interaction between dietary CP level and lysine intake, shown in Fig. 14.2, represents a

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"D

Lysine intake (mg day-1)

Fig. 14.2. Daily weight gain and lysine intake of chicks fed diets containing 140 (•), 160 (o), 180 (▲), 200 (A), 220 (■), 240 (□), 260 (♦) and 280 (O) g crude protein kg 1. (From D'Mello, 1988; source of data: Morris etal., 1987. Reproduced with permission of The World's Poultry Science Association.)

Lysine intake (mg day-1)

Fig. 14.2. Daily weight gain and lysine intake of chicks fed diets containing 140 (•), 160 (o), 180 (▲), 200 (A), 220 (■), 240 (□), 260 (♦) and 280 (O) g crude protein kg 1. (From D'Mello, 1988; source of data: Morris etal., 1987. Reproduced with permission of The World's Poultry Science Association.)

reassessment by D'Mello (1988) of the original data published by Morris et al. (1987). In this study, the summit diet containing 280 g CP kg-1, first-limiting in lysine, was diluted with a basal diet containing 140 g CP kg-1 of identical amino acid balance and metabo-lizable energy (ME) content to yield a series of diets varying in CP content and first-limiting in lysine. Each diluted diet was supplemented with graded levels of pure lysine which, in theory, should have produced responses compatible with those obtained merely by diluting the summit diet. However, it is readily seen (Fig. 14.2) that the growth response obtained on dilution is quite distinct from that obtained on supplementation of each diluted diet with pure lysine. The displacement of response curves at each CP level and the appearance of discrete responses to lysine supplementation question the rigour of the initial procedures used to validate the diet-dilution technique. The CP effect on growth responses to amino acids also holds for tryptophan (Abebe and Morris, 1990b) and methionine (Morris et ai, 1992).

Discrepancies recorded between the two techniques are not just restricted to growth responses. As will be seen later in this chapter, marked differences also occur in carcass fat contents of chicks on diluted and amino acid supplemented regimes.

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